Enterovirus subtyping in a routine UK laboratory setting between 2013 and 2017

Howson-Wells, Hannah C.; Winckles, Stephen; Aliker, Camille; Tarr, Alexander W.; Irving, William L.; Clark, Gemma; McClure, C. Patrick

Abstract

© 2020 Elsevier B.V. Background: Human enteroviruses (EV) are the leading cause of viral meningitis. EV genotyping is predominantly performed through amplification and sequencing of viral capsid protein-1 (VP1), frequently by national reference laboratories (NRLs). Objective: To determine the frequency of genotyping failure in our NRL-submitted samples and apply a superior alternative assay to resolve untyped specimens. Study design: We initially audited genotyping data received for a cohort of patients in the East Midlands, UK by the NRL between 2013 and 2017, then identified an alternative RT-PCR typing method by literature review and evaluated primers from both assays in silico against comprehensive publicly available genomic data. The alternative assay was further optimised and applied to archived nucleic acids from previously untypable samples. Results: Genotyping data showed a significant increase in untypable EV strains through the study period (p = 0.0073). Typing failure appeared unrelated to sample type or viral load. In silico analyses of 2,201 EV genomes showed high levels of mismatch between reference assay primers and clinically significant EV-species, in contrast to a selected alternative semi-nested RT-PCR VP1-typing assay. This alternative assay, with minor modifications, successfully genotyped 23 of 24 previously untypable yet viable archived specimens (EV-A, n = 4; EV-B, n = 19). Phylogenetic analyses identified no predominant strain within NRL untypable isolates, suggesting sub-optimal reference assay sensitivity across EV species, in agreement with in silico analyses. Conclusion: This modified highly sensitive RT-PCR assay presents a suitable alternative to the current English national reference VP1-typing assay and is recommended in other settings experiencing typing failure.

Citation

Howson-Wells, H. C., Winckles, S., Aliker, C., Tarr, A. W., Irving, W. L., Clark, G., & McClure, C. P. (2020). Enterovirus subtyping in a routine UK laboratory setting between 2013 and 2017. Journal of Clinical Virology, 132, Article 104646. https://doi.org/10.1016/j.jcv.2020.104646

• Journal Article Type: Article
• Acceptance Date: Sep 14, 2020
• Online Publication Date: Sep 17, 2020
• Publication Date: Nov 1, 2020
• Deposit Date: Sep 28, 2020
• Publicly Available Date: Sep 18, 2021
• Journal: Journal of Clinical Virology
• Print ISSN: 1386-6532
• Electronic ISSN: 1873-5967
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 132
• Article Number: 104646
• DOI: https://doi.org/10.1016/j.jcv.2020.104646
• Keywords: Enterovirus; VP1; Genotyping; Viral diagnostics; RT-PCR; Reference laboratory
• Public URL: https://nottingham-repository.worktribe.com/output/4924767
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S1386653220303887
• Additional Information: This article is maintained by: Elsevier; Article Title: Enterovirus subtyping in a routine UK laboratory setting between 2013 and 2017; Journal Title: Journal of Clinical Virology; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.jcv.2020.104646; Content Type: article; Copyright: © 2020 Elsevier B.V. All rights reserved.